The present invention is directed to a method for inhibiting either iNOS or COX-2, or both in mammals using flavone compounds. The present invention is also directed to a method of activating K+ channels in mammals; as well as methods for treating septic shock, treating or preventing aneurysm, inhibiting expression of angiotensin converting enzyme and reducing inflammation and related pathological changes using these compounds. Presently preferred compounds are oroxylin A (5,7-dihydroxy-6-methoxy flavone) and wogonin (5,7-dihydroxy-8-methoxy flavone).
Septic shock and multiple-organ failure are catastrophic consequences of an invasive infection. Septic shock has been estimated to occur in more than 500,000 cases per year in the United States alone. Septic shock is the most common cause of death in non-coronary intensive care units. As more antibiotic-resistant strains of bacteria evolve, the incidence of septic shock is expected to increase. Overall mortality rates from septic shock range from 30% to 90%. Aggressive antibiotic treatment and timely surgical intervention are the main therapies, but in many cases are insufficient. The search for new drug therapies has not been successful. For example, only small, but not statistically significant improvements in 28-day mortality compared to placebo was found when the compound Deltibant was administered to human patients suffering systemic inflammatory response syndrome and presumed sepsis (R. Stone, J. Am. Med Assoc., vol. 277, pp. 482-487 (1997)).
Lipopolysaccharide (LPS) is believed to be the principal agent responsible for inducing sepsis syndrome, which includes septic shock, systemic inflammatory response syndrome, and multi-organ failure. Sepsis is a morbid condition induced by a toxin, the introduction or accumulation of which is most commonly caused by infection or trauma. The initial symptoms of sepsis typically include chills, profuse sweating, irregularly remittent fever, prostration and the like; followed by persistent fever, hypotension leading to shock, neutropenia, leukopenia, disseminated intravascular coagulation, acute respiratory distress syndrome, and multiple organ failure.
LPS, also know as endotoxin, is a toxic component of the outer membrane of Gram-negative microorganisms (e.g., Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa). Compelling evidence supports the toxic role of LPS; all pathophysiological effects noted in humans during Gram-negative sepsis can be duplicated in laboratory animals by injection of purified LPS. The mechanism by which LPS activates responsive cells is complex and not fully understood. The host response to Gram-negative bacterial infection depends on effector cell recognition of the bacteria, LPS, or both and involves both serum proteins and cell membrane receptors. When bacteria and LPS are removed via endocytosis and phagocytosis by reticuloendothial cells, concomitant activation of the host immune response by LPS results in the secretion of cytokines by activated macrophages, which in turn can trigger the exaggerated host responses associated with septic shock.
The normal immune response begins when neutrophils squeeze through the blood-vessel walls searching for bacterial pathogens in the surrounding tissue. Neutrophils can kill bacteria directly by releasing toxic chemicals or enzymes, such as elastase or collagenase. The neutrophils also attract other leukocytes to the area, including lymphocytes, macrophages, and monocytes, the last two of which release powerful immune-response activators called cytokines. The cytokines, in turn, stimulate more immune cell activity and increase the number of cells coming to the area by making the blood-vessel wall more permeable. Then, as the number of bacteria decreases, other cytokines signal to bring the normal immune response to an end.
If the cutoff mechanism fails, however, sepsis can begin. In sepsis, humoral and cellular mediators cascade in a process that becomes at least temporarily independent of the underlying infection. Excess neutrophils and macrophages are drawn to the site of infection, releasing excess immune-stimulating cytokines, eventually triggering the release of substances that damage the blood-vessel wall. More monocytes and macrophages come to the site and release more cytokines. Eventually, the blood vessels are so damaged and leaky that blood pressure falls and the blood can no longer supply nutrients to the body""s organs. Entire organs can begin to shut down. Many patients die after losing the function of two or more organs.
Two cytokines that play an important role in sepsis are interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF alpha). These two polypeptides can raise body temperature, increase the expression for adhesion molecules on neutrophils and endothelial cells (promoting adhesion of leukocytes), stimulate the production of vasodilating prostaglandins (thus increasing the permeability of blood vessels), trigger the release of other cytokines, stimulate neutrophils, and activate fibroblasts. All these processes enhance the probability of organ failure seen in severe septicemia. Drug therapies that target only one of these two cytokines have proved ineffective (See Stone). Drug therapies that are effective against general inflammatory responses have not proven to be effective against the cascading acute inflammation that produces septicemia. There is a need for drugs that can inhibit this cascading system at the beginning steps of production of IL-1 and TNF alpha.
Other important cytokines, chemokines, and other proteins having proinflammatory activity include interferon-gamma (IFN gamma), interleukin-6 (IL-6), macrophage chemotactic protein (MCP), inducible nitric oxide synthetase (iNOS), mitogen-activated protein kinases (MAPKs), macrophage inflammatory protein, KC/CINC (growth related gene), tissue factor (TF), granulocyte-macrophage-colony stimulating factor (Gm-CSF) and phosphotyrosine phosphatase (PTPase).
Prostaglandins are also involved in the proinflammatory response; e.g., prostaglandins increase the permeability of the blood-vessel wall. Cyclooxygenase (COX; prostaglandin endoperoxide synthase) catalyzes the conversion of arachidonic acid to prostaglandin (PG) endoperoxide (PGH2), which is the rate limiting step in prostaglandin biosynthesis. Two isoforms of COX have been cloned from animal cells: the constitutively expressed COX-1, and the mitogen-inducible COX-2. Prostaglandins produced as a result of the activation of COX-1 may have physiological functions such as the antithrombogenic action of prostacyclin released by the vascular endothelium, and the cytoprotective effect of PGs produced by the gastric mucosa. However, COX-2 is the enzyme expressed following the activation of cells by various proinflammatory agents including cytokines, endotoxin and other mitogens. These observations suggest that COX-2 instead of COX-1 may be responsible for inducing production of the prostaglandins involved in inflammation. Only a few pharmacological agents that suppress the expression of COX-2 without affecting COX-1 have been identified, for example, glucocorticoids and radicicol. However, these agents have undesirable side effects.
There is a need for compounds that selectively inhibit COX-2, and that act as potent anti-inflammatory agents, with minimal side effects. To prevent septicemia, such a compound should also inhibit the production of a wide variety of proinflammatory cytokines, especially TNF alpha and IL-1, chemokines, and protein-tyrosine kinases.
Nitric Oxide (NO) was originally identified in vascular endothelial cells (Palmer et al. (1987) Nature 327:524-526 and Palmer et al.(1988) Nature 333:664-666) and has been identified as being identical to endothelium-derived relaxing factor (Moncada et al. (1989) Biochem. Pharmacol. 38:1709-1715; Furchgott (1990) Acta Physiol. Scand. 139:257-270 and Iganarro (1990) Annu. Rev. Phamacol. Toxicol. 30:535-560). Besides endothelial cells, NO formation has been demonstrated in macrophages (Hibbs et al. (1987) Science 235:473-476 and Marletta et al. (1988) Biochemistry 27:8706-8711), neutrophils (McCall et al. (1989) Biochem. J. 262:293-297; Salvemini et al. (1989) Proc. Natl. Acad. Sci. USA 86:6328-6332 and Wright et al. (1989) Biochem. Biophys. Res. Commun. 160:813-819), some tumor cells (Amber et al. (1988) J. Leuk. Biol. 44:58-65), adrenal glands (Palacios et al. (1989) Biochem. Biophys Res. Commun. 165:802-809). Kupffer cells (Billiar et al. (1989) J. Exp. Med. 169:1467-1472) and in brain tissue (Garthwaite et al. (1988) Nature 336:385-388; Knowles et al. (1989) Proc. Natl. Acad. Sci.USA 86:5159-5162 and Bredt and Snyder (1989) Proc. Natl. Acad. Sci. USA 86:9030-9033).
Endothelium-derived NO relaxes the smooth muscles of blood vessels (Palmer et al. (1987) Nature 327:524-526 and Ignarro et al. (1987) Proc. Natl. Acad. Sci. USA 84:9265-9269) and inhibits platelet adhesion (Radomski et al. (1987) Biochem. Biophys. Res. Commun., 148:1482-1489). NO production by cocultures of Kupffer cells and hepatocytes mediates inhibition of hepatocyte protein synthesis (Billar et al. (1989) J. Exp. Med. 169:1467-1472). NO is responsible for mediating the cytotoxic effects of macrophages and neutrophils (Hibbs et al. 91987) J. Immunol. 138:550-556). NO has also been shown to be a major neuronal messenger in the brain (Bredt and Snyder (1989) Proc. Natl. Acad. Sci. USA 86:9030-9033). The mediation of functions of tissues as diverse as the brain, endothelium and blood cells indicates a wide-spread role for NO as a messenger molecule.
NO is formed by nitric oxide synthetase (NOS) from L-arginine with stoichiometric formation of L-citrulline. Studies have shown that a guanidino nitrogen of L-arginine is used to form NO (Iyengar et al. (1987) Proc. Natl. Acad. Sci. USA 84:6369-6373; Palmer et al. (1988) Nature 333:664-666 and Marietta et al. (1988) Biochemistry 27:8706-8711).
The formation of NO appears to involve the same or similar enzyme in brain and endothelial cells but a different enzyme in macrophages. The brain-endothelium enzyme has been found to require calcium and calmodulin for activity (Bredt and Snyder (1990) Proc. Natl. Acad. Sci. USA 87:682-685). The macrophage enzyme does not require calcium-calmodulin but does require tetrahydrobiopterin for activity (Tayeh and Marietta (1989) J. Biol. Chem. 264:19654-19658; Soo Kwon et al. (1989) J. Biol. Chem. 264:20496-20501).
The brain (i.e., calmodulin-dependent) NOS enzyme has been purified to homogeneity from rat brain, revealing a 150,000 kD protein (Bredt and Snyder (1990) Proc. Natl. Acad. Sci. USA 87:682-685).
In addition to the differences between NOS activities in brain and endothelial cells as compared to macrophages, the regulation of NOS expression appears to differ as well. The synthesis of NO does not occur in macrophages unless they have been exposed to endotoxin (e.g., bacterial lipopolysaccharide) or cytokine (e.g., interferon-gamma, -beta or alpha, tissue necrosis factor-alpha or -beta). However, in the brain and vascular endothelium, NOS is present without exposure to inducing agents (Knowles et al. (1990) Biochem. J. 270:833-836). The arginine derivative L-N-omega-nitroarginine (NO2Arg) has been described as being a competitive inhibitor of NOS (Moore (1990) Br. J. Pharmacol. 99:408-412).
NO has been demonstrated to mediate neuronal relaxation of intestines (Bult et al. (1990) Nature 345:346-347; Gillespie et al. (1989) Br. J. Pharmacol. 98:1080-1082 and Ramagopal and Leighton (1989) Eur. J. Pharmacol. 174:297-299) and to mediate stimulation by glutamate of cGMP formation (Bredt and Snyder (1989) Proc. Natl. Acad. Sci. USA 86:9030-9033). Glutamate, the major excitatory neurotransmitter in the brain, acts through several receptor subtypes, some of which stimulate the formation of cGMP (Ferrendelli et al. (1974) J. Neurochem. 22:535-540). Glutamate, acting at N-methyl-D-aspartate (NMDA) subtype of receptors, is responsible for neurotoxic damage in vascular strokes. Selective antagonists of NMDA glutamate receptors prevent neuronal cell death in animal models of hypoxic-ischemic brain injury (Choi (1990) J. Neurosci. 10:2493-2501). Glutamate neurotoxicity has also been implicated in neurodegenerative disorders such as Alzheimer""s and Huntington""s diseases (Choi (1990) J. Neurosci. 10:2493-2501 and Meldrum and Garthwaite (1990) Trends in Pharmacol. Sci. 11:379-387).
As decribed above, nitric oxide (NO) has been shown to be an important regulatory molecule in diverse physiological functions such as vasodilation, neural communication and host defense. Molecular cloning and sequencing analysis have revealed the existence of at least three main types of NOS isoforms. NOS is present in the vascular endothelium (eNOS); in central and peripheral neurons (nNOS); and is also constitutive (cNOS). Activation is Ca+2-dependent. Continuous release of NO by cNOS keeps the vasculature in an active state of vasodilation. Various agonists such as bradykinin and acetylcholine have been shown to trigger cNOS-mediated NO production through increasing intracellular Ca+2. NOS in macrophages and hepatocytes, on the other hand, is inducible (iNOS) and its activation is Ca+2-independent (Duval et al., Mol. Pharmacol. 50: 277-84, 1996, Yuan, T., Febs. Lett. 431:210-4, 1998). After exposure to endotoxin and/or cytokines, iNOS can be induced in various cells such as macrophages, Kupffer cells, smooth muscle cells and hepatocytes. The induced iNOS catalyzed the formation and release of a large amount of NO, which play a key role in the pathophysiology of a variety of diseases including septic shock (Pedoto, A. et al., Crit. Care Med. 26:2021-8, 1998). NO production catalyzed by iNOS therefore may reflect the degree of inflammation and provides a measure by which effects of drugs on the inflammatory process can be assessed.
It is known that inhibition of either iNOS or COX-2 enzymes prevents aneurysm (Miralles, M. et al. J. Vasc. Surg. May 1999, Vol. 5, pp. 884-892; and Fukuda, S. Circulation, May 2000, 101 (21) pp. 2532-2538).
Expression of cyclooxygenase-2 (COX-2) in various tissue preparations following LPS treatment has also been reported (Quan, N. et al., Brain Res. 802:189-197; Lee, S. H. et al., J. Biol. Chem. 267: 25934-25938, 1992). This enzyme also is considered to play a major role in inflammatory process by catalyzing the production of prostaglandins.
Compounds which inhibit iNOS or COX-2 would be useful anti-inflammatory agents as has been described above; and a compound which inhibits or prevents induction of both enzymes at the same time should be particularly useful. To date, compounds which inhibit both enzymes have not been identified.
Therefore, one object of the invention is to identify anti-inflammatory agents. A further object of the invention is to identify compounds which inhibit induction of both iNOS and COX-2.
Four types of K+ channels have been described in vascular and nonvascular smooth muscle. These are: (1) calcium-activated (2) voltage-dependent (also called delayed rectifier) (3) ATP-sensitive and (4) inwardly rectifying K+ channels. Calcium-activated K+ channels (KCa channels) have been found in virtually every type of smooth muscle. These K+ channels are activated by increasing levels of intracellular calcium. They may also be activated by membrane depolarization, although this mechanism also requires calcium at physiologic membrane potentials. Calcium-activated K+ channels are thought to be the most abundant in vascular smooth muscle, with up to 104 channels estimated to be present per cell (Nelson and Quayle, xe2x80x9cPhysiological Roles and Properties of Potassium Channels in Arterial Smooth Musclexe2x80x9d, Am. J. Physiol. 268 (4Pt 1): C799-822, 1995).
One of the important physiological roles of KCa channels is regulation of smooth muscle or myogenic tone. Elevation of intravascular pressure depolarizes smooth muscle cells in resistance arteries and causes vasoconstriction. This tone has been referred to as xe2x80x9cmyogenic tonexe2x80x9d and is a major contributor to peripheral resistance. KCa channels play an important role in the control of myogenic tone. It has been proposed that pressure-induced membrane depolarization and increases in intracellular Ca2+ activate KCa channels. Activation of KCa channels would increase K+ efflux, which would counteract the depolarization and constriction caused by pressure and vasoconstrictors. Activation of KCa channels acts as a negative feedback mechanism to limit vasoconstriction.
KCa channels are regulated by endogenous vasoactive substances. Most vasoconstrictors (e.g. norepinephrine, angiotensin II, endothelin, and serotonin) depolarize vascular smooth muscle. It is conceivable that inhibition of KCa channels contributes to this membrane depolarization. Recently, angiotensin II and a thromboxane A2 agonist (U-46619) have been shown to inhibit KCa channels from coronary artery smooth muscle. Muscarinic receptor stimulation has been shown to inhibit KCa channels in airway and colonic smooth muscle. (Faraci and Sobey, xe2x80x9cRole of Potassium Channels in Regulation of Cerebral Vascular Tonexe2x80x9d, J. Cereb. Blood Flow Metab. 18 (10): 1047-63, 1998).
Activation of KCa channels would tend to hyperpolarize smooth muscle and lead to muscle relaxation. xcex2-Adrenergic stimulation activates KCa channels in airway smooth muscle cells and thus may contribute to xcex2-adrenergic bronchodilation. This activation of KCA channels in airway and coronary artery smooth muscle cells appears to be caused by phosphorylation mediated by an adenosine 3xe2x80x2,5xe2x80x2-cyclic monophosphate (cAMP)-dependent protein kinase as well as a direct G protein pathway. Recent evidence indicates that guanosine 3xe2x80x2,5xe2x80x2-cyclic monophosphate (cGMP)-dependent protein kinase can also activate KCA channels in smooth muscle cells isolated from cerebral and coronary arteries. Nitric oxide can activate cGMP-dependent protein kinase through stimulation of guanylyl cyclase and elevation of cGMP. Furthermore, nitric oxide has also been reported to directly activate KCA channels in aortic smooth muscle. Vasorelaxation of some vascular beds (e.g., mesenteric and cerebral arteries) in response to nitric oxide appears to involve activation of KCA channels.
Like calcium-activated K+ channels, voltage-dependent K+ channels are activated in response to membrane depolarization, but this process occurs independent of the intracellular calcium concentration. Because both voltage-dependent and calcium-activated K+ channels are activated by depolarization, 4-aminopyridine (4-AP) can be used to distinguish responses mediated by either channel. Tetraethylammonium (TEA) is a poor inhibitor of voltage-dependent K+ channels unless very high concentrations are used. The estimated number of voltage-dependent K+ channels per cell in arteries is about 103.
Compared with other K+ channels, much less is known about the functional importance of voltage-dependent K+ channels. It has been suggested that activity of voltage-dependent K+ channels influence resting cerebral vascular tone. These K+ channels are also activated by increases in arterial blood pressure. Recent studies suggest that activation of voltage-dependent K+ channels may contribute to mechanisms that produce cerebral vasorelaxation in response to NO and endothelium-derived hyperpolarizing factor (EDHF) (Faraci and Sobey, 1998).
There are three physiological roles of Kv channels which include: (1) Repolarization of the action potential. Despite the wide distribution of Kv channels, relatively few studies have been conducted on the physiological role of this channel in arterial smooth muscle. Because the channel is activated by depolarization, it may be involved in action potential repolarization in electrically excitable smooth muscle preparations such as the portal vein, and this is a principal function of the channel in other excitable cells, including neurons and cardiac muscle. However, most arteries generally respond to stimuli with graded membrane potential changes, and therefore Kv channels are unlikely to be involved in action potential repolarization in these arteries. (2) Regulation of the membrane potential. Kv channels provide an important K+ conductance in the physiological membrane potential range in arteries that do not generate action potentials. Activation of Kv channels by membrane depolarization, e.g., in response to pressurization or vasoconstrictors, may limit membrane depolarization. Kv channels may also be directly modulated by vasoconstrictors and vasodilators, and a 4-AP-sensitive K+ current is inhibited by a histamine H1 receptor agonist in coronary arteries. It was suggested that inhibition of the 4-AP-sensitive current occurred as a result of increased intracellular Ca2+ concentration through intracellular C2+ release. A related observation is that intracellular Mg2+ (10 mM) inhibits Kv currents positive to xe2x88x9215 mV in arterial smooth muscle cells. (3) hypoxic pulmonary vasoconstruction. Pulmonary arteries constrict in hypoxia, which minimizes blood perfusion in poorly ventilated areas of the lung. This hypoxic vasoconstriction contrasts with the hypoxic vasodilation seen in many small systemic arteries and which may involve an activation of other types of K+ channels. During hypoxia, pulmonary arteries depolarize and may generate action potentials. The resulting pulmonary vasoconstriction is abolished by removal of extracellular Ca2+ and by Ca2+ channel antagonists such as verapamil, suggesting that Ca2+ entry through voltage-dependent Ca2+ channels is important in the hypoxic response.
Recent studies suggest a role for K+ channels in hypoxia-induced membrane depolarization and constriction. K+ channel inhibitors such as TEA+ and 4-AP increase tone in isolated pulmonary vessels and increase perfusion pressure in the isolated perfused lung. Thus K+ channels contribute to the membrane potential in pulmonary arteries as they do in systemic arteries. Because K+ channels regulate the membrane potential of pulmonary smooth muscle, hypoxia may depolarize by inhibiting K+ channels. It has recently been directly shown that hypoxia inhibits voltage-activated K+ currents in these arteries. The voltage dependence of the hypoxia-sensitive channel suggests that it is a member of the Kv or KCa families. A number of mechanisms have been proposed to link hypoxia to channel inhibition. KCa channels in rat pulmonary arterial myocytes are activated by intracellular ATP. Therefore a fall in intracellular ATP during hypoxia may inhibit this channel. However, the ATP connection in smooth muscle cells is generally well conserved during hypoxia. Cellular redox status has also been proposed as the link between hypoxia and K+ channel activity, and an increase in cellular reducing agents causes inhibition of K+ channels in pulmonary arteries.
One key characteristic of ATP-sensitive K+ channels (KATP) is that their activity may reflect the metabolic state of the cell. These K+ channels are sensitive to intracellular ATP, which inhibits channel activity. Dissociation of ATP from the channel results in channel opening and membrane hyperpolarization. Other metabolically related stimuli, including reductions in PO2 or pH, also open the channel and produce vasorelaxation. It is estimated that a few hundred ATP-sensitive K+ channels are present per cell in arteries. The number is much less than that for calcium-activated K+ channels.
KATP channels have several physiological roles. The channel is activated by a number of vasodilators, and the associated membrane hyperpolarization causes part of the resulting vasodilation in many cases. The KATP channel may also be inhibited by vasoconstrictors which would tend to cause depolarization and constriction. The channel is involved in the metabolic regulation of blood flow; it is activated in conditions of increased blood demand, e.g., in hypoxia, either by release of vasodilators from the surrounding tissue or as a direct result of hypoxia on the vascular smooth muscle cells. Finally, the channel may be active in the resting state, because inhibition of KATP channels can lead to increased resistance to blood flow in some vascular beds.
Inwardly rectifying K+ channels (KIR channels) are present in a variety of excitable and nonexcitable cells, including some arterial smooth muscle cells. The name of this channel comes from the observation that the membrane potential is controlled, e.g., by voltage clamp of the cell, inward currents through the KIR channel (movement of K+ from the extracellular solution into the cell) are larger than outward currents. This is because the KIR channel is activated by membrane hyperpolarization, in contrast to KV and KCa channels, which are activated-by membrane depolarization.
Although outward currents through the KIR channel are small, in most physiological situations the cell membrane potential is positive to the EK, providing an electrochemical gradient for K+ to leave the cell. The KIR channel therefore normally conducts an outward hyperpolarizing membrane current. From a physiological standpoint, these small outward currents are therefore of considerable interest. Outward K+ movement through the cardiac muscle KIR channel is limited by voltage-dependent channel closure on membrane depolarization and may also involve block of outward current through the channel by intracellular Mg2+. However, the role of intracellular Mg2+ is complex because channels that are blocked are unable to undergo voltage-dependent closure.
The physiological roles of the KIR channel in cells other than smooth muscle include regulating the resting membrane potential, preventing membrane hyperpolarization to values more negative than the Ek by the electrogenic Na+-K+-ATPase, and minimizing cellular K+ loss and therefore energy expenditure during sustained membrane depolarization. The roles of the KIR channel in arterial smooth muscle are incompletely understood but may include some of the functions such as mediates K+-induced dilations and regulation of membrane potential.
In summary, activation of K+ channels in arterial smooth muscle cells can increase blood flow and lower blood pressure through vasodilation. Inhibition of K+ channels in arterial smooth muscle leads to vasoconstriction. Four types of K+ channels (KV, KCa, KATP and KIR channels) have been identified to regulate the membrane potential of vascular and nonvascular smooth muscle cells.
KCa channels in arterial smooth muscle cells respond to changes in intracellular Ca2+ to regulate membrane potential. KCa channels appear to play a fundamental role in regulating the degree of intrinsic tone of resistance arteries. These channels help regulate arterial responses to pressure and vasoconstrictors.
KV channels regulate membrane potential in response to depolarizing stimuli, and these channels may be involved in hypoxia-induced membrane depolarization in the pulmonary vasculature.
KATP channels are targets of a number of vasodilating stimuli, including hypoxia and adenosine. A variety of antihypertensive drugs (e.g., minoxidel sulfate, diazoxide, lemakalim, pinacidil) act through activation of KATP channels. Pathological conditions such as hypotension associated with septic shock may involve excessive activation of KATP channels.
KIR channels appear to mediate external K+-induced hyperpolarizations and dilations of resistance arteries and thus provide a mechanism for linking the metabolism of surrounding cells (e.g., neurons) to blood flow.
All of these K+ channel types may be involved in the actions of a variety of vasodilators and vasoconstrictors, and their function may be altered in diseases. K+ channels in arterial smooth and nonvascular smooth muscle (such as uterine and pulmonary) muscle are important modulators of blood vessel diameter, and muscle tone. Our results indicate that oroxylin A is a Ca2+xe2x80x94 activated K+ channel opener, but is not a KATP channel opener. Preliminary results further indicate that oroxylin A may activate other K+ channels such as KV or KIR channels.
The present invention is directed to methods for inhibiting expression of iNOS, COX-2, or both using a flavone and pharmaceutically acceptable salts thereof. The present invention is also directed to a method for activation of potassium channels by flavones; a method for treating septic shock with flavones; a method for inhibiting expression of angiotensin converting enzyme with flavones; a method for reducing inflammation and related diseases with flavones; and a method for treating or preventing aneurysms with flavones.
More particularly, the present invention is directed to the use of compounds of the formula I 
wherein p is an integer of zero to five;
R1, at each occurrence, is independently selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkoxy, alkenoxy, alkynoxy, thioalkoxy, aliphatic acyl, CF3, CN, NO2, OH, NH2, CHxe2x95x90NOH, SO2xe2x80x94(C1-C3 alkyl), SO3xe2x80x94(C1-C3 alkyl), N(C1-C3 alkyl)-CO(C1-C3 alkyl), C1-C3alkylamino, alkenylamino, alkynylamino, di(C1-C3 alkyl)amino, COOH, C(O)Oxe2x80x94(C1-C3 alkyl), C(O)NHxe2x80x94(C1-C3 alkyl), C(O)N(C1-C3 alkyl)2, haloalkyl, alkoxylcarbonyl, alkoxyalkoxy, carboxaldehyde, carboxamide, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, aroyl, aryloxy, arylamino, biaryl, thioaryl, heterocyclyl, heterocycloyl, alkylaryl, aralkyl, alkylheterocyclyl, heterocyclylalkyl, sulfonamido, carbamate, aryloxyalkyl and C(O)NH(benzyl);
R2, R4 and R6 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkoxy, alkenoxy, alkynoxy, thioalkoxy, aliphatic acyl, CF3, CN, NO2, OH, NH2, CHxe2x95x90NOH, SO2xe2x80x94(C1-C3 alkyl), SO3xe2x80x94(C1-C3 alkyl), N(C1-C3 alkyl)-C(O)(C1-C3 alkyl), C1-C3alkylamino, alkenylamino, alkynylamino, di(C1-C3 alkyl)amino, haloalkyl, alkoxylcarbonyl, alkoxyalkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, aroyl, aryloxy, arylamino, biaryl, thioaryl, alkylaryl, aralkyl, sulfonyl, heterocyclyl, heterocycloyl, alkylheterocyclyl, heterocyclylalkyl, sulfonamido, halogen and aryloxyalkyl; and
R3 and R5 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, alkenyl, alkynyl, aryl, aralkyl, biaryl, heterocyclyl, heterocycloyl, alkylheterocyclyl, heterocyclylalkyl, cyanomethyl, cycloalkyl, cycloalkenyl and cycloalkylalkyl;
wherein R1, R2, R3, R4, R5 and R6 are unsubstituted or substituted with at least one electron donating or electron withdrawing group;
and pharmaceutically acceptable salts thereof in the methods described above. Presently preferred flavones are 5,7-dihydroxy-6-methoxy flavone (oroxylin A, wherein p is zero, R2, R3, R5 and R6 are hydrogen, and R4 is methoxy for formula I above) and 5,7-dihydroxy-8-methoxy flavone (wogonin, wherein p is zero, R2, R3, R4 and R5 are hydrogen, and R6 is methoxy for formula I above). For the preferred flavones, when p=zero, the phenyl ring (substituted by (R1)p in formula I) is unsubstituted. The unsubstituted phenyl ring is defined herein either as when p is five and R1 is hydrogen, or when p=0. Useful derivatives of the compounds of Formula I include esters, carbamates, animals, amides, optical isomers and pro-drugs thereof.
For the practice of any aspect of this invention, a bactericidal amount of an antibiotic may be co-administered with the flavone.